Proper completion of DNA replication is critical to preserve genomic integrity. The DNA replication machinery unwinds and copies parental DNA strands, generating highly dynamic replication fork structures. Blockage of a moving replication fork by obstacles, such as DNA lesions and naturally occurring DNA-protein complexes, can lead to fork damage, which ultimately can result in the accumulation of genomic instability and cancer. In order to maintain the integrity of forks, cells are equipped with an evolutionary conserved surveillance mechanism called the replication checkpoint. In humans, this checkpoint pathway is initiated by the phosphatidyl-inositol-3-kinase-like kinase ATR. While it is well established that ATR is critical to maintain the integrity of replication forks, how this kinase promotes fork stabilization and repair remains unclear. Important clues regarding the roles of the replication checkpoint came from studies conducted in Saccharomyces cerevisiae (budding yeast). The high level of conservation of this pathway and the power of yeast genetics makes the budding yeast an ideal model organism to dissect the molecular mechanisms by which replication checkpoint signaling coordinates fork repair. Recent work by this applicant revealed that Mec1 (the yeast ortholog of human ATR) mediates a crucial interaction between the fork protein Dpb11 and the Rtt107-Slx4 scaffolds, which together form a platform for the coordination of DNA repair factors, including endonuclease, ubiquitin-ligase and sumo-ligase complexes. Based on these findings, this proposal will explore a model where Mec1 promotes fork repair by coordinating the spatiotemporal recruitment of repair factors at damaged forks via assembly of the Rtt107-Slx4-Dpb11 complex. First, the recruitment of the Rtt107-Slx4- Dpb11 complex and associated repair factors to damaged forks will be examined using live cell microscopy. Second, a targeted genetic screen will be conducted to elucidate the involvement of other pathways with the repair function of the Rtt107-Slx4-Dpb11 complex. Finally, mass spectrometry-based proteomic techniques will be used to investigate the role of the Rtt107-Slx4-Dpb11 complex in sumoylation mediated fork repair. For the purpose of these experiments, rigorous training will be obtained on how to acquire and process microscopy and mass spectrometry data. Also, comprehensive training in the field of genetic will be gained through course work in eukaryotic genetics. Taken together, the goal of the proposed study is to expose the mechanistic action of the replication checkpoint in fork stabilization and repair. Results generated here will help understand how cells can survive in the presence of fork damaging agents such as camptothecin analogues, cisplatin and hydroxyurea, which are commonly used in cancer treatment. This knowledge could provide important insights for the design of more effective chemotherapeutic strategies.